DK159100B - PROCEDURE FOR PREPARING A LIQUID VIRAL VACCINE CONTAINING A STABILIZER AND STABILIZER THAT CAN BE USED IN SUCH A PROCEDURE - Google Patents

Description

DK 159100 B

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The present invention relates to a method for producing a liquid viral vaccine and, in particular, to a stabilizer for use in the preparation of such a vaccine.

5 Due to the worldwide distribution of vaccines and disparate ambient temperatures, there has been a need to stabilize these preparations for transport and use. Several stabilization methods have been used in the past.

10 a) Low temperatures (-10 to -70 ° C).

The need for low temperature storage facilities, which are not always available, limits the practical implementation of this measure.

b) Lyophilization.

Although lyophilization suffers from the disadvantage of being a costly measure, lyophilized vaccines are reasonably stable and stored at 4 to 8 ° C until used. However, during this storage period, the vaccines slowly deteriorate until after approx. For 12 to 24 months, 20 does not have a sufficient titre to confer immunity. Furthermore, since the lyophilized vaccine must be reconstituted before use, the liquid reconstituted preparation loses potency at room temperature. This can result in insufficient titres to confer immunity and result in a failure of the immunization program.

(c) Stabilizers.

Stabilizers are chemical compounds added to the vaccine and are used in conjunction with either low temperature storage or lyophilization. Chemical stabilizers, e.g. SPGA (a stabilizer described by Bovamick et al.,

J. Bact. 59: 509-522 (1950)) and the like are described in the prior art literature. As described by Bovarnick et al., 1 liter of SPGA contains 0.218 M

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2 sucrose (74.62 g), 0.00376 Μ KE ^ PO ^ (0.52 g), 0.0071 M i ^ HPO ^ (1.25 g), 0.0049 M potassium glutamate (0.912 g) and 1 % serum albumin (10 g). Various modifications of SPGA are known with respect to the indicated quantities 5 and constituents, and it is easy for those skilled in the art to modify this stabilizer. Thus, potassium glutamate is often replaced with sodium glutamate while the modified formulations are still referred to as SPGA. U.S. Patent No. 3,783,098, corresponding to French Patent No. 2,115,379, cites e.g. to an SPGA stabilizer containing monosodium glutamate instead of monocalcium glutamate (column 6, lines 5-11). U.S. Patent No. 3,933,585 discloses an SPGA stabilizer which, at liter of sterile distilled water contains 74.62 g of sucrose, 0.45 g of KI 2 PO 2, 1.35 15 g of 1 HPO 2, 0.956 g of monosodium L-glutamate and 40 ml of a 25% * solution of albuminosol (human albumin).

In general, an SPGA stabilizer contains approx.

2 to approx. 10% sugar, e.g. sucrose, ca. 0.05 to approx. 0.3% of a mono- or dibasic alkali metal phosphate salt or a mixture of such salts, e.g.

KE ^ PO ^, l ^ HPO ^, Na ^ PO ^, and / or Na2HPO ^, ca. 0.05 to approx. 0.2% glutamic acid alkali metal salt, e.g. sodium or potassium glutamate, and approx. 0.5% to 2% serum albumin, e.g. bovine serum albumin or human albumin. Various components substitutions can be made in the formulation of the SPGA stabilizer. Eg. For example, sucrose as described in U.S. Patent No. 3,783,098 (column 3, lines 59-61) may be partially or completely replaced with a starch hydrolyzate, e.g. glucose or dextran, 30 and albumin can be replaced, in whole or in part, with casein or polyvinylpyrrolidone, as described in U.S. Patent No. 3,783,098 (column 3, line 8) and U.S. Patent No. 3,915,794, respectively. None of the prior art stabilizers, nor those described in Chemical 35 Abstracts 8_0 (1974), 41031 f., U.S. Pat.

4,147,772, and Belgian Patent No. 665076 3

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contains both L-arginine and monosodium glutamate or glutamic acid. These stabilizers are therefore not permanently stable.

It is therefore an object of the present invention to provide an improved method for preparing a liquid vaccine by mixing an inactivated or attenuated virus and a stabilizer consisting essentially of 1.5 to 2.1 parts by weight of partially hydrolyzed gelatin with a molar weight of approx. 3000, 10 7.0 to 13.0 parts by weight of monosaccharide, disaccharide or sorbitol, 0.4 to 0.6 parts by weight of an in vitro cell culture medium, and such amount of a physiologically acceptable acid buffer capable of maintaining pH of 6.0 to 6.5.

The process according to the invention is characterized in that the stabilizer contains 0.35 to 0.7 parts by weight of L-glutamic acid and 0.75 to 1.3 parts by weight of L-arginine.

The invention also relates, as mentioned, to a stabilizer for use in the preparation of a liquid vaccine, as mentioned above, which essentially consists of 1.5 to 2.1 parts by weight of partially hydrolyzed gelatin having a mole weight of approx. 3000, 7.0 to 13.0 parts by weight of monosaccharide, disaccharide or sorbitol, ca. 0.4 to 0.6 parts by weight of an in vitro cell culture medium and such amount of physiologically acceptable acidic buffer effective to adjust the pH of 6.0 to 6.5.

The stabilizer of the invention is characterized in that it also contains 0.35 to 0.7 parts by weight of L-glutamic acid and 0.75 to 1.3 parts by weight of L-arginine.

Such a stabilizer provides increased sustained stability which none of the known stabilizers exhibit.

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The liquid vaccine can be obtained by thawing a frozen vaccine or by reconstituting a lyophilized vaccine. Examples of live viruses are measles, mumps, rubella, chickenpox, polio and hepatitis, or a combination of two or more of the viruses mentioned.

Hydrolyzed gelatin is used to obtain a soluble, non-gelling protein matrix with little or no pyrogenicity or antigenicity.

By partially hydrolyzed gelatin is meant gelatin which has been subjected to a partial hydrolysis to obtain partially hydrolyzed gelatin having a molecular weight of approx. 3000. This gelatin hydrolysis product has approximately the same amino acid composition as gelatin. Unlike gelatin which forms gels but is insoluble in cold water, hydrolyzed gelatin does not form gel but is soluble in cold water and other common liquids such as milk and orange juice. Aqueous solutions containing up to approx. 10% hydrolyzed gelatin does not significantly increase viscosity.

At over approx. 10% concentration increases viscosity slowly. 20 At approx. At 50% concentration, the solutions are quite viscous.

The typical amino acid composition for hydrolyzed gelatin is as follows:

Alanine 8.5%

Arginine 7> 9% Aspartic acid 5.7%

Cystine 0.08%

Glutamic Acid 9.5%

Glycine 22.8%

Histidine 0.77% Hydroxyproline 13-14%

Isoleucine 1.3%

Leucine 2.9%

Lysine 4.2%

Methionine 0.78% Phenylalanine 2.0%

Proline 13.8% 5

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Serine 3.3%

Threonine 1.9%

Tyrosine 0.40%

Valine 2.4% Partially hydrolyzed gelatin can be obtained by enzymatic hydrolysis of gelatin by a proteolytic enzyme such as papain, chymopapain and bromelain, although other known hydrolysing agents may be used, e.g. acid hydrolysis. Appropriate hydrolyzed gelatin is obtained from Wilson and Co., Inc., 10 Calumet City, Illinois under the trade name SOL-U-PRO.

The stabilizer also contains a monosaccharide, e.g. sorbitol, or a disaccharide, e.g. sucrose, lactose or maltose. Sucrose is preferred.

The L-glutamic acid can be used as such or in the form of its 15. sodium salt, monosodium glutamate.

The L-arginine can be used as such or in the form of the hydrochloride.

The acidic buffer may be any physiologically acceptable buffer which maintains the desired pH of 6 to 6.5, e.g. a phosphate buffer, acetate buffer or citrate buffer. A phosphate buffer is preferred. The stabilizer is diluted from 3 to 8 times, preferably approx. 5.5 crange, its own weight of distilled water before use.

A cell culture medium is understood to mean a nutrient medium that enables cell growth in vitro. Special nutritional media are e.g. Medium 199, Morgan et al., Proc. Soc. Exp. Biol.

& Med., 73: 1-8, 1950; Basal Medium Eagle, Eagle, Science, 122, 501-504, 1955; In Vitro, Volume 6, No. 2, 1970; Dul-becco's Modified Eagle's Medium, Dulbecco et al., Virology, 30 8, 396, 1959; Smith et al., J. Virol., 12, 185-196, I960;

In Vitro, Volume 6, No. 2, 1970; Minimum Essential Medium (Eagle), Science, 130, 432 (1959) and RPMI Media, Moore et al., 199, 519-524, 1967; In Vitro, Volume 6, No. 2, 1970.

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The stabilizer according to the invention can be used for the preparation of various liquid viral vaccines, e.g. vaccines against measles, mumps, rubella, respiratory syncytial virus, parainfluenza types 1, 5 2 and 3 and cytomegalovirus.

The composition of the stabilizer according to the invention can e.g. be as follows:

Ingredient Weight parts / 50 ml 10 Partially hydrolyzed gelatin 1.5 - 2.1

Mono- or disaccharide 7.0 -13.0 Nutrient medium (solid) 0.4 - 0.6 L-glutamic acid 0.35- 0.7 L-arginine 0.75-1.3 Physiologically acceptable buffer to adjust pH to 6.0

to 6.5 0.05 to 0.2 M

Particularly preferred formulations for the liquid viral vaccine stabilizer as contemplated by the present invention are as follows:

Partially hydrolyzed gelatin y} g g

Sucrose or sorbitol 10.0 g

Medium 199 0.55 g

Monosodium glutamate 0.5 g L-arginine HCl 1.0 g 2.5

Sodium pho sphatpuf f is 1 M, pH 6.2 5 ml

Water to 50.0 ml

In addition, the stabilizer may contain, and contains forego, a small amount of NaHCO 3 and phenol red. In a composition such as the one described above, NaHCO 3 may be present in an amount of approx. 1.2 g and phenol red in an amount of approx. 0.01 g. Although special preparations are described in it

DK 159100 B

7 above, it will be understood that the ratios and concentrations of each component may be varied. A volume of mass vaccine is usually diluted by approx. 2 to approx. 12 volume stabilizer.

The following examples further explain the present invention.

Example 1 40 ml of measles virus concentrate, which has been stored at -70 ° C, is thawed in a water bath at 25 ° C and then kept at 4 -10 8 ° C. The liquid virus concentrate is divided into two aliquots of 16.5 ml each.

A) An aliquot of 16.5 ml of this viral fluid is diluted with 50 ml of the following stabilizer, which is sterilized by passage through a 0.2 µm membrane.

15 M phosphate buffer, pH 6.2 5.0 ml

Sorbitol, 25% aqueous solution 7.1 ml

Hydrolyzed gelatin, 25% ts aqueous solution 7.1 ml

Medium 199 30.7 ml

Formulation is performed under aseptic conditions and laminar flow cap. To avoid microbial growth, add neomycin (0.1 ml, 2500 units) to the preparation. The diluted vaccine is placed in 2 ml vials (0.7 ml vaccine per ampoule), which are immediately flame-sealed and stored at 37 ° C.

B) The second aliquot of 16.5 ml is treated as the first except that the stabilizer preparation further contains 0.5 g monosodium glutamate, 1.0 g L-arginine HCl and 10 g sucrose instead of sorbitol.

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Storage stability of the vaccines is shown in the following table:

Titers ^ of liquid vaccines stored at 37 ° C

5 Time, Stabilizer with Stabilizer with

hours formulation A formulation B

O 3.9 3.7 24 2.3 3.1 48 2.0 2.7 10 72 1.5 2.5 ^ titers are expressed as TCID ^ q / 0.1 ml.

Example 2 25 ml of measles virus concentrate, which has been stored at -70 ° C, is thawed in a water bath at 25 ° C and then kept at 4 -8 ° C. The liquid viral concentrate is diluted with 75 ml of Stabilizer B (Example 1) and placed in 2 ml vials. The ampoules are heat sealed and stored at 2 - 8 ° C. The storage stability of the vaccine is described in the following table:

Stability of liquid vaccine stored at 2 - 8 ° C

20

Time, months Titer (TCIDr-0 / 0.1 ml) 0 3.7 1 4.1 l 3.1 £ 3.5 25 ® 3.3

Example 3

The following table compares the stability of 4 potions of liquid measles vaccine prepared at various times using stabilizers of composition A or B as described in Example 1.

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Titre (TCID 50 / 0.1 ml) after the specified number of hours Stabilizer _ at 37 ° C_ 0 24 48 72 5 1 A 3.9 2.3 2.0 1.5 1 B 3.7 3.1 2, 7 2.5 2 A 4.0 3.1 2.5 1.8 2 B 3.9 3.6 3.0 2.6 3 A 3.9 - 2.2 2.1 10 3 B 3.6 3.3 3.0 2.5 4 A 3.5 2.1 1.6 1.5 4 B 3, '6 - - 3.5 -' - 3.1 2.4

Claims (10)

A process for preparing a liquid vaccine in which an inactivated or attenuated virus and a stabilizer comprising essentially 5 of 1.5 to 2.1 parts by weight of partially hydrolyzed gelatin having a molecular weight of 3000, 7.0 to 13 are mixed. , 0 parts by weight of monosaccharide, disaccharide or sorbitol, 0.4 to 0.6 parts by weight of an in vitro cell culture medium, and such amount of a physiologically acceptable acid buffer capable of maintaining a pH of 6.0 to 6.5, characterized in that it also contains 0.35 to 0.7 parts by weight of L-glutamic acid and 0.75 to 1.3 parts by weight of L-arginine. Process according to claim 1, characterized in that the disaccharide is sucrose. Process according to claim 1, characterized in that the buffer is a phosphate buffer. Method according to claim 1, characterized in that the virus is measles, mumps, rubella, chickenpox, polio or hepatitis, herpes simplex 20 type 1 or herpes simplex type 2 virus or combinations thereof. Process according to claim 1, characterized in that the stabilizer consists essentially of 1.8 parts by weight of partially hydrolyzed gelatin with a molecular weight of 3000, 10.0 parts by weight of monosaccharide, disaccharide or polyol, 0.5 parts by weight of an in vitro cell culture medium. , 0.5 parts by weight of L-glutamic acid, 1.0 parts by weight of L-arginine and such amount of physiologically acceptable acidic buffer effective for adjusting the pH of 6.0 to 6.5. Process according to claim 5, characterized in that the disaccharide is sucrose. A stabilizer for use in the manufacture of a liquid vaccine according to claim 1, consisting essentially of 1.5 to 2.1 parts by weight of partially hydrolyzed gelatin having a molecular weight of 3000, 7.0 to 13.0 parts by weight of monosaccharide, disaccharide or sorbitol, 0.4 to 0.6 parts by weight of an in vitro cell culture medium and such amount of physiologically acceptable acid buffer effective to adjust the pH of 6.0 to 6.5, characterized in that it also 10 contains 0.35 to 0.7 parts by weight of L-glutamic acid and 0.75 to 1.3 parts by weight of L-arginine. Stabilizer according to claim 7, characterized in that the disaccharide is sucrose. Stabilizer according to claim 7, characterized in that it consists essentially of 1.8 parts by weight of partially hydrolyzed gelatin with a molecular weight of 3000, 10 parts by weight of a monosaccharide, disaccharide or sorbitol, 0.5 parts by weight of an in vitro cell culture medium. , 0.5 parts by weight of L-glutamic acid, 1.0 parts by weight of L-arginine and such an amount of a physiologically acceptable acidic buffer effective to adjust the pH of 6.0 to 6.5. Stabilizer according to claim 9, characterized in that the disaccharide is sucrose.